Bioengineered periosteal progenitor cell sheets to enhance tendon-bone healing in a bone tunnel

Tendon-bone tunnel healing is crucial for long term success in anterior cruciate ligament (ACL) reconstruction. The periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondroblasts during tendon-bone healing. We developed a scaffold-free method using polymerized fibrin-coated dishes to make functional periosteal progenitor cell (PPC) sheets. Bioengineered PPC sheets for enhancing tendon-bone healing were evaluated in an extra-articular bone tunnel model in rabbit. PPC derived from rabbit tibia periosteum, cultivated on polymerized fibrin-coated dishes and harvested as PPC sheet. A confocal microscopy assay was used to evaluate the morphology of PPC sheets. PPC sheets as a periosteum to wrap around hamstring tendon grafts were pulled into a 3-mm diameter bone tunnel of tibia, and compared with a tendon graft without PPC sheets treatment. Rabbits were sacrificed at 4 and 8 weeks postoperatively for biochemical as-say and histological assay to demonstrate the enhancement of PPC sheets in tendon-bone healing. PPC spread deposit on fibrin on the dish surface with continuous monolayer PPC was ob-served. Histological staining revealed that PPC sheets enhance collagen and glycosaminoglycans deposition with fibrocartilage formation in the tendon-bone junction at 4 weeks. Collagen fiber with fibrocartilage formation at tendon-bone junction was also found at 8 weeks. Matured fibrocartilage and dense collagen fiber were formed at the tendon-bone interface at 8 weeks by Masson trichrome and Safranin-O staining. Periosteal progenitor cell monolayer maintains the differentiated capacity and osteochondral potential in order to promote fibrocartilage formation in tendon-bone junction. Bioengineered PPC sheets can offer a new feasible therapeutic strategy of a novel approach to enhance tendon-bone junction healing.